Craniofacial studies in chicken embryos confirm the pathogenicity of human FZD2 variants associated with Robinow syndrome

ABSTRACT Robinow syndrome is a rare disease caused by variants of seven WNT pathway genes. Craniofacial features include widening of the nasal bridge and jaw hypoplasia. We used the chicken embryo to test whether two missense human FZD2 variants (1301G>T, p.Gly434Val; 425C>T, p.Pro142Lys) were sufficient to change frontonasal mass development. In vivo, the overexpression of retroviruses with wild-type or variant human FZD2 inhibited upper beak ossification. In primary cultures, wild-type and variant human FZD2 significantly inhibited chondrogenesis, with the 425C>T variant significantly decreasing activity of a SOX9 luciferase reporter compared to that for the wild type or 1301G>T. Both variants also increased nuclear shuttling of β-catenin (CTNNB1) and increased the expression of TWIST1, which are inhibitory to chondrogenesis. In canonical WNT luciferase assays using frontonasal mass cells, the variants had dominant-negative effects on wild-type FZD2. In non-canonical assays, the 425C>T variant failed to activate the reporter above control levels and was unresponsive to exogenous WNT5A. This is the first single amino acid change to selectively alter ligand binding in a FZD receptor. Therefore, FZD2 missense variants are pathogenic and could lead to the altered craniofacial morphogenesis seen in Robinow syndrome.

FZD2 425C>T codes for P142L and FZD2 1301G>T codes for G434V.(A) The predicted ribbon structure of FZD2 protein created in PyMOL software using AlphaFold (Identifier# AF-Q14332-F1).The location of affected amino acids Proline 142 (P142 red spheres) in the cysteine rich domain (cyan) and Glycine 434 (G434 royal blue spheres) in the 5 th transmembrane domain (TMD) (orange) are shown.The C-terminus is represented by red.(B') Magnified view of the Proline at position 142 (red spheres) (B) replaced by Leucine (green spheres).(C') Magnified view of Glycine at position 434 (blue spheres) (C) replaced by Valine (pink spheres).(D) Representation of the known functional domains of FZD2 (green, red) and the dishevelled interaction domain (turquoise) are illustrated.The predicted location of the ADRS missense mutations p.Pro142Leu (c.425C>T) and p.Gly434Val (c.1301G>T).The number of patients classified as ADRS patients are shown with black dots (White et al., 2018;Zhang et al., 2022).

Fig. S2. Skeletal preparation of additional embryos injected with GFP, wild-type hFZD2 or mutant hFZD2 variants
Embryos injected GFP, wild-type hFZD2, hFZD2 425C>T , or hFZD2 1301G>T into the frontonasal mass at stage 15 (E2.5) and fixed 10 days post-injection at stage 38.(A-L) Wholemount skulls cleared and stained with alcian blue (cartilage) and alizarin red (bone).(A-C) GFP virus allowed normal patterning and ossification of frontonasal mass derived bones including premaxillary, nasal and prefrontal.(D-L) Embryos injected with wild-type hFZD2 or mutant hFZD2 viruses had no defects in the upper beak morphology but had missing nasal bone on the injected side (white dashed lineright nasal bone) while the nasal bone was present on the left side (yellow dashed line).The prefrontal bone (green dashed line) was absent in >90% of embryos and the premaxillary bone (white dashed line) was present but showed faint alizarin red staining.The size of the premaxilla appeared to be smaller compared to GFP controls.(K, L) Some embryos injected with hFZD2 1301G>T had beak shortening and deviation (6/19).(L) An embryo with unossified skull and short upper beak injected with hFZD2 1301G>T .The faint cartilage stain (blue) in all skulls is a technical error.Key: ln, left nasal; pmx, premaxilla; prf, prefrontal; rn, right nasal.Scale bar: A-L= 2mm.S12 for a detailed list of embryos analyzed at stage 28.We also show examples of embryos with lower GAGstaining (red cross) that were not used for molecular analysis.Scale bar = 500µm

Fig. S3 .
Fig. S3.Analysis of missing bones in embryos injected with human FZD2 viruses Contingency analysis (Fisher's exact test) showing total embryos with normal or missing (A) nasal (B) Premaxillary, or (C) Prefrontal bones at stage 38 in vivo.

Fig. S4 .
Fig. S4.Embryos at stage 28 (E5.5)injected with GFP, wt hFZD2 or hFZD2 variants Paraffin-embedded serial sections of stage 28 embryos stained with anti-GAG antibody (green fluorescence) and counter-stained with Hoechst (blue) to detect the viral spread in the frontonasal mass.Embryos injected with (A-F) GFP (G-L) wt hFZD2, (M-R) 425C>T, (S-X) 1301G>T viruses Note: Sections shown in S4A, G, M, S are exactly the same sections as in Fig. 2A-D respectively.We reproduced the images so that all specimens in the study are represented.Please refer to TableS12 for

Fig. S5 .
Fig. S5.qRT-PCR analysis showing effects of hFZD2 viruses on mediators of the WNT pathway and skeletogenic mediators RNA-isolated from day 6 or day 8 micromass cultures, three biological replicates per virus type containing pool of 8-9 micromass cultures per biological replicate.The expression of each biological replicate was normalized to 18s RNA and then these ΔCt values were used to calculate the ΔΔCt relative to the average levels of expression of the gene in GFP.The graphs showing gene expression changes on (A) day 6 and (B) day 8.All statistical analysis was done using one-way ANOVA, Dunnett's multiple comparison test in GraphPad Prism 10.1.0.The error bars represent one standard deviation.Genes showing statistically significant differences are shown in red text.Black dashed line represents control GFP.Key: Each black dot represents a biological replicate.The p values are displayed with red asterisk on the graph.* -p < 0.05.

Fig. S6 .
Fig. S6.qRT-PCR analysis showing effects of hFZD2 viruses on mediators of the WNT pathway and skeletogenic mediators RNA-isolated from day 6 or day 8 micromass cultures, three biological replicates per virus type containing pool of 8-9 micromass cultures per biological replicate.The expression of each biological replicate was normalized to 18s RNA and then these ΔCt values were used to calculate the ΔΔCt relative to the average levels of expression of the gene in GFP.The graphs showing gene expression changes on (A) day 6 and (B) day 8.All statistical analysis was done using one-way ANOVA, Dunnett's multiple comparison test in GraphPad Prism 10.1.0.The error bars represent one standard deviation.Genes showing statistically significant differences are shown in red text.Black dashed line represents control GFP.Key: Each black dot represents a biological replicate.The p values are displayed with red asterisk on the graph.* -p < 0.05.

Fig. S7 .
Fig. S7.Immunostaining on DF1 cells infected with high titer hFZD2 virus and quantification of viral titer (A, A') Immunocytochemistry performed on DF1 chicken fibroblasts infected with 2µl of high titre viral stock were fixed at 36h post-infection.The cells were stained with anti-GAG antibody (virus) and counterstained with Hoechst.(B) Quantification of viral titer showed that all hFZD2 viruses had higher than the recommended titer of 1 x 10 7 IU/mL for performing overexpression studies.The error bars represent one standard deviation.Scale bar = 200μm.Key: IU -Infectious units, ns -not significant

Fig. S8 .
Fig. S8.The specificity of the DSHB nuclear β-catenin was tested in western blots loaded with chicken cell lysate.DF1 chicken fibroblasts transfected with proviral plasmids containing either GFP or wild-type human DVL1.Cells were cultured for 4 weeks before lysis.Blot probed with anti-nuclear β-catenin (DSHB, PY489) at 0.5 µg/ml.There is a major band at ~86 kDa coinciding with the expected molecular weight of chicken β-catenin from UniProt.The lower bands are likely nonspecific.

Table S3 . Analysis of upper beak skeleton at stage 38 (E12.5) skulls stained in wholemount
1 -Total embryos analyzed for skeletal phenotypes caused by the FZD2 viruses 2-Total number of embryos with external phenotypes including short or deviated upper beak observed externally 3 -The total number of samples with a phenotype was analyzed in detail and classified as embryos with missing bones or faint or absent alizarin red staining in bones derived from the frontonasal mass Key: n; nasal bone, N; total sample size, pmx; premaxillary bone, prf; prefrontal bone, -missing bone, therefore ossification changes are not quantified